Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components that participate in what has been referred to as the coagulation “cascade” are proenzymes or zymogens, enzymatically inactive proteins that are converted to proteolytic enzymes by the action of an activator, which is itself an activated clotting Factor. Coagulation factors that have undergone such a conversion are generally referred to as “active factors”, and are designated by the addition of a lower case “a” suffix (e.g., Factor VIIa).
Activated Factor X (“Xa”) is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, or pathways, that promote the activation of Factor X. The “intrinsic pathway” refers to those reactions that lead to thrombin formation through utilisation of factors present only in plasma. A series of protease-mediated activations ultimately generates Factor IXa, which, in conjunction with Factor VIIIa, cleaves Factor X into Xa. An identical proteolysis is effected by Factor VIIa and its co-Factor, tissue factor, in the “extrinsic pathway” of blood coagulation. Tissue factor is a membrane bound protein and does not normally circulate in plasma. Upon vessel disruption, however, it can complex with Factor VIIa to catalyse Factor X activation or Factor IX activation in the presence of Ca2+ and phospholipid. The relative importance of the two coagulation pathways in haemostasis is still unclear.
Factor IXa (FIXa) is a trypsin-like serine protease that serves a key role in haemostasis by generating, as part of the Xase complex, most of the Factor Xa required to support proper thrombin formation during coagulation (reviewed in Hoffman M. and Monroe D. M., III (2001) A cell-based model of hemostasis. Thromb Haemost 85, 958-965). Congenital deficiency of Factor IXa activity is the cause of the X-linked bleeding disorder haemophilia B affecting approximately 1:100,000 males. These haemophilia patients are currently treated by replacement therapy with either recombinant or plasma-derived coagulation Factor IX.
Factor IX is a vitamin K-dependent coagulation factor with structural similarities to Factor VII, Factor X, and protein C. The circulating zymogen form, which has a plasma half-life of about 18-30 hours, consists of 415 amino acids divided into four distinct domains comprising an N-terminal γ-carboxyglutamic acid rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. Activation of Factor IX occurs by limited proteolysis at Arg145-Ala146 and Arg180-Val181 releasing a 35-aa fragment, the so-called activation peptide (Schmidt A. E. and Bajaj S. P. (2003) Structure-function relationships in Factor IX and Factor IXa. Trends Cardiovasc Med 13, 39-45). The activation peptide is heavily glycosylated containing two N-linked and up to four O-linked glycans.
γ-Carboxyglutamic acid (Gla) is a unique amino acid that binds to calcium. It is a modified form of glutamic acid (Glu) and can be produced in vivo by the post-translational modification of glutamate residues. Carboxylation of glutamic acid in this way enables calcium binding and allows the attachment of proteins such as procoagulants and anticoagulants to phospholipids. This enzyme-mediated reaction, known as γ-carboxylation (gamma carboxylation), requires vitamin K as a cofactor.
Some mature proteins contain a domain that is rich in amino acids that have been converted to γ-carboxyglutamic acid in this way. This is known as a GLA domain. This GLA domain is often responsible for the high-affinity binding of calcium ions by the protein. Such a GLA domain may be found in a variety of different proteins. For example, blood coagulation Factors VII, IX and X and prothrombin all include a GLA domain that comprises a number of Gla amino acid residues.
Van Cott et al (1996) Journal of Molecular Recognition 9, 407-414 describes affinity purification of biologically active and inactive forms of recombinant Human Protein C.
Full gamma-carboxylation of the 12 Glu residues in the Gla domain of FIX in recombinant production represents a major challenge. FIX produced in Chinese hamster ovary (CHO) cells shows a specific activity of FIX of approx. 50%, an activity level which correlates with the degree of gamma-carboxylation of FIX expressed from the cell line. There is therefore a need to develop a downstream separation method to remove FIX species with suboptimal gamma-carboxylated Gla domain.